The search for agents which can effectively act against fungi and mycoses injurious to plants and animals in a suitably selective manner such that the host plant or animal is not significantly harmed can be an expensive and time-consuming process. A significant degree of trial and error normally is required in order to ascertain even potential candidates among a wide variety of possibilities, and thereafter to further determine whether those with potential in fact exhibit the requisite activity and selectivity suitable for commercial use. Much advantage could be realized were it possible to provide a relatively rapid and precise method for screening a wide variety of possible agents based upon some specific characteristic or property useful as a predictor of anti-fungal activity and fungal specificity. Apart from the evident economic advantages, a method of this type would enable larger universes of possible agents to be screened, with corresponding increased potential for identifying particularly suitable fungicides. This is particularly important as increasing numbers of injurious fungi are becoming resistant to commercially available fungicides (Koeller, W., and Scheinpflug, H., Plant Disease, 71: 1066-1074 (1987)).
As discussed in detail hereinafter, the present invention provides a screening process which is based upon the finding that chemical and biological agents having fungicidal activity can be identified based upon their ability to inhibit spindle pole body formation and/or function in yeast strains that otherwise overproduce spindle pole bodies.
The accurate segregation of chromosomes during mitosis is essential for normal eukaryotic cell division, and precisely organized complex microtubule arrays are required during this process so that reproduction is faithful. Chromosome segregation is mediated by the mitotic spindle apparatus which comprises the spindle microtubules, the kinetochore (a structure attaching spindle fibers to chromosomes), and the spindle pole, which organizes the microtubules. In mammals and some other organisms, the spindle pole equivalent is the centrosome; it comprises the centrosphere and a pair of centrioles. In yeast and other fungi, the spindle pole is called the spindle pole body; it is permanently associated with the nuclear envelope because the nuclear envelope does not disassemble during yeast mitosis.
Spindle pole bodies in yeast not only function in the formation of the mitotic spindle, but also serve crucial roles in karyogamy (fusion of gametic nuclei), where they mediate the initiation of nuclear fusion within the zygotes and in the formation of spore walls (Baum, P., et al., Yeast Cell Biology, Alan R. Liss, Inc., New York, 1986, pages 151-158). Microtubules originating from the yeast spindle pole body have been implicated in other cellular processes, including bud formation and/or growth (Snyder, M., and Davis, R. W., Cell 54: 743-754 (1988)). Therefore, it is essential that the behavior of yeast spindle pole bodies be highly integrated with other cellular functions.
Whereas the mammalian centriole is defined microscopically as a roughly spherical symmetrical cytoplasmic structure, the fungal spindle pole body is a planar structure embedded in the nuclear membrane. Plant cells have no functional equivalent of a spindle pole body visible by microscopy. Besides these differences in morphology and cellular location, the existence of agents with differential action on fungal and mammalian microtubules such as griseofulvin and benomyl argues for differences in the mitotic apparatus in these cell types.
Cellular division in yeast, especially common baker's yeast Saccharomyces cerevisiae, has been studied for decades, and the genetics of this organism is very well developed (see Mortimer, R. K., and Schild, D., Microbiol. Rev49: 181-212 (1985), for example, for a genetic map). Having a low DNA content, short generation times, ease of cultivation and preservation, simplicity of nutritional requirements and rapid growth, yeast is readily amenable to genetic analysis and manipulation using classical and molecular techniques.
At least four genetic loci have been described which affect spindle pole body synthesis in yeast. One is a cell division cycle (cdc) mutant, cdc31, which uncouples spindle pole body duplication from other aspects of cell division (Baum, et al., cited above). Spindle pole body duplication fails to occur despite the occurrence of budding and DNA replication.
Rose, et al., have characterized a gene implicated in mutations that prevent karyogamy, denoted KAR1and shown that it is required for both mitosis and conjugation (Rose, M. D., and Fink, G. R., Cell 48: 1047-1060 (1987)). The KARl product is apparently required for mitosis, spindle body duplication, and the assembly of both intranuclear and extranuclear microtubules. Overproduction of the gene product blocks spindle pole body duplication. Mutant strains show defects both in spindle body duplication and chromosome disjunction. Both mitotic and mating mutant cells have defects associated with the spindle plaque. Cells arrested in mitosis by either insufficient or excess KAR1 gene product are unable to duplicate spindle plaque, and loss of KAR1 function results in abnormally long extranuclear microtubules. Microtubules of abnormal length are also found in kar1-1 cells during the mating process.
Baum, et al., identified a gene denoted ESP1 for extra spindle pole bodies which functions in the duplication of the spindle pole body (Baum, et al., cited above, and Baum, P., et al., Mol. Cell. Biol. 8: 5386-5397 (1988)). The investigators created a number of mutant strains, including one having a temperature-sensitive lethal mutation that deregulated spindle pole duplication. At permissive temperatures (.about.23.degree. C.), the mutant grew normally. At restrictive temperatures (above 34.5.degree. C.; 36.degree. C. was used in most experiments reported), growth, as evidenced by DNA synthesis and cell division, was arrested, and the cells continued to increase their number of spindle pole bodies incessantly. Large numbers of spindle pole bodies accumulated in the yeast, an effect that was lethal to most of the cells.
Snyder and Davis (cited above) identified and cloned a spindle pole body gene using an immunoscreening technique and denoted it SPA1 for spindle pole antigen. The gene product, which is overproduced in esp1-1 mutants and cofractionates with the yeast nuclear envelope, apparently is a protein associated with the yeast spindle pole. Mutant strains exhibit a high frequency of chromosome nondisjunction, abnormal spindles, and chromosome mis-segregation. A mutational analysis indicates the gene product to be important for cell growth, chromosome segregation, and other cellular processes.